Proteolytic activity is responsible for damage to connective tissues and basement membranes as a complication of the inflammatory and/or immune response and other disease processes, such as cancer cell invasion and metastasis. The inflammatory response contributes, for example, to the pathological changes in a number of acute and chronic processes involving diverse organs and tissues such as the lungs, bone, heart, joints, skin and periodontium, etc.
The proteinases involved in these responses or disease processes include matrix metalloproteinase (MMP's), MMP-like proteinases and related proteinases, serine proteinases and other proteinases. The MMP's are zinc and calcium-dependent for hydrolytic cleavage of substrate proteins and are secreted or released by a variety of host cells (e.g., polymorphonuclear neutrophils (PMN's), macrophages, bone cells, epithelium and fibroblasts). Certain other genetically distinct MMP's called membrane-type MMP's (MT-MMP's) are cell membrane-bound; others are secreted into the extracellular matrix (ECM). With serine proteinases, the amino acid serine acts as a nucleophile for hydrolytic cleavage of substrate protein. Serine proteinases are released, e.g., by triggered leukocytes, more specifically by the azurophilic granules of PMN's, and other cells including malignant tumor cells.
Several studies have shown that the expression and activities of MMPs are pathologically elevated over the body's endogenous anti-proteinase shield in a variety of diseases such as cancer metastasis, rheumatoid arthritis, multiple sclerosis, periodontitis, osteoporosis, osteosarcoma, osteomyelitis, bronchiectasis, chronic pulmonary obstructive disease, skin and eye diseases. Proteolytic enzymes, especially MMPs, are believed to contribute to the tissue destruction damage associated with these diseases.
Some metalloproteinases (MMP's) and their association with diseases are discussed by M. E. Ryan, et al., Curr. Op. Rheum., 1996, 8:238-247. More than twenty MMP's have been identified and the number is growing. These include Interstitial Collagenases MMP-1 (fibroblast-type), MMP-8 (polymorphonuclear leukocyte-PMNL-type or collagenase-2), MMP-13 (collagenase-3); Gelatinases MMP-2 (72-kD gelatinase A) and MMP-9 (92-kD gelatinase B); Stromelysins MMP-3 (stromelysin-1), MMP-10 (stromelysin -2), and MMP-7 (matrilysin or putative metalloproteinase (PUMP) -1); Membrane Type (MT-MMP's), MMP-14 (MT.sub.1 -MMP), MMP-15 (MT.sub.2 -MMP), MMP-16 (MT.sub.3 -MMP); others are, for example, MMP-11 (stromelysin-3), MMP-12 (macrophage metalloelastase) and MMP-20. Enamelysin (MMP-20) is described by Llano et al., Biochem. 1997, 36:15101-15108, and can also be expressed by human cancer cells such as squamous carcinoma cells of the human tongue indicating its potential contribution to cancer progression and invasion (Salo et al., J Dent. Res. 1998, 77:829, Abstr. No. 1978). Related proteinases include TACE's and ADAM's fertilin or meltrin (metalloproteinase/disintegrin).
MMP's, MMP-like and related proteinases such as TACE's, ADAM's, etc., are involved in processing and modification of molecular phenomena such as tissue remodeling (Birkedal-Hansen, Current Opin. Cell Biol. 1995, 7:728-735; J F Woessner, Jr., FASEB J 1991, 5:2145-2154), cytokine actions (S. Chandler et al., J Neuroimmunol. 1997, 72:155-161), cell-cell fusion (R H van Huijsduijen, Gene 1998, 206:273-282; Huovila et al., Curr. Opin. Cell Biol. 1996, 8:692-699; Yagami-Hiromasa et al., Nature 1995, 377:662-656), angiogenesis, growth factor actions, integrin and other adhesion factors and their receptor processings. See also, A. C. Perry et al., Biochem. Biophy Acta 1994, 1207:134-137. The ADAM enzymes are membrane proteins with A Disintegrin and Metalloproteinase Domain (Wolfsberg et al., Dev. Biol. 1995, 169:378-383). TACE is tumor necrosis factor converting enzyme.
MMP-like proteinases and related proteinases are metalloproteinases distinct from classic MMP's and can be involved in cellular processing of pro-TNF alpha (Tumor Necrosis Factor), cellular shedding of cytokine receptors, adhesion molecules, etc. as described by S. Chandler et al., J Neuroimmunol. 1997, 72:155-161. MMP's and MMP-like and related enzymes, e.g., ADAM's, TACE's, etc., also mediate the release of TNF alpha (Watanabe et al., Eur. J Biochem. 1998, 253: 576-582) and are involved in membrane-bound processing of TNF alpha by monocytes induced by bacterial-virulence factors. This event is mediated by membrane-bound metalloproteinases. Shapira et al., J. Period Res. 1997, 32:183-185.
There is extensive evidence for the association between proteinases and a large number of disease processes. Microbial proteinases can act in concert with host proteinases in the promotion of tissue destruction as seen in periodontium (Sorsa et al., Infect. Immun. 1992, 60: 4491-4495). Recent studies indicate that a serine protease, i.e., elastase, may play a role in connective tissue breakdown and tissue invasion in the Dunning rat model of cancer invasion and metastases (prostate cancer) (Lowe and Isaacs, Cancer Res. 1984, 44:744-52). Also involved in initiating the proteinase cascade that mediates tumor invasion and metastasis are trypsin and chymotrypsin-like activity (Sorsa et al., J. Biol. Chem. 1997, 272:21067-21074). Serine proteinase is expressed in human cancers such as ovarian carcinoma and cholangiosarcoma (Sorsa et al., J. Biol. Chem. 1997, 272:21067-21074).
The role of MMP's has been well-established in a great many disease states, e.g., tumor invasion and metastasis (Stetler-Stevenson et al., Annu. Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys. Acta 1987, 907:191-217) and bone and cartilage degradation (Greenwald et al., Bone 1998, 22:33-38; Ryan et al., Curr. Op. Rheumatol. 1996, 8;238-247). MMP-20 is expressed by oral squamous cell carcinoma cells (Salo et al., J. Dent. Res. 1998, 77:829, Abstr. No. 1978). Bourguignon et al. (Mol. Biol. Cell. 1997, 8i Supplement, Abstract 1603) describe the association of metalloproteinase with matrix degradation as being responsible for promoting lymphocyte infiltration that destroys insulin-producing pancreatic islet cells. Cytokines (TNF alpha) and MMP's have also been implicated in the pathogenesis of multiple sclerosis (Liedtke et al., Ann. Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol.1997, 72:155-71). MT.sub.1 -MMP has been found to act in the growth and spread of breast cancer cells (Li et al., Mol. Carcinog. 1998, 22:84-89).
There are many other disorders in which extracellular protein degradation/destruction plays a prominent role. Examples of such diseases include osteoporosis, arthritides, acquired immune deficiency syndrome (AIDS), burns, wounds such as bed sores and varicose ulcers, fractures, trauma, gastric ulceration, skin diseases such as acne and psoriasis, lichenoid lesions, epidermolysis bullosa, aphthae (reactive oral ulcer), dental diseases such as periodontal diseases, peri-implantitis, jaw cysts and other periapical cysts, dental conditions which are the target of root canal treatment or endodontic treatment, related diseases, external and intrinsic root resorption, caries etc.
The serine proteinases include human leukocyte elastase (HLE) and cathepsin G, and additional serine proteinases are involved in the cascade of pathways involved in connective tissue breakdown including but not limited to, plasmin, plasminogen activator, tumor-associated trypsins, etc.
MMP's and serine proteinases can work in combinations to bring about destruction of most of the elements of the extracellular matrix and basement membranes. As examples of the major interaction between MMP's and serine proteinases during tissue breakdown, 1) cathepsin G can activate MMP-8; 2) the serine proteinase Human Leukocyte Elastase (HLE) can inactivate TIMP's, the major endogenous Tissue Inhibitors of Matrix Metalloproteinases, 3) MMP-8 and MMP-9 can activate .alpha..sub.1 -Proteinase Inhibitor (.alpha..sub.1 -PI), the major endogenous inhibitor of human leukocyte elastase, (S. K. Mallya, et al., Annuals of the New York Academy of Science, 1994, 732:303-314) and 4) tumor-associated-trypsin-2 can efficiently activate latent pro MMP's (Sorsa et al., J. Biol. Chem. 1997, 272:21067-21074).
Tetracyclines, including chemically modified tetracyclines, can inhibit MMP-mediated tissue breakdown in vitro and in vivo, in part by binding to metal ions (calcium or zinc) in the MMP molecular structure. See, e.g., R. F. Zernicke et al., Journal of Rheumatology, 1997, 24:1324-31; T. Sorsa et al., Journal of Rheumatology, 1998, 25:975-82; Golub et al., Adv. Dental Research 1998, in press.
Certain tetracyclines have been shown to suppress matrix metalloproteinases independently of tetracycline antibiotic activity. U.S. Pat. Nos. 5,459,135 to Golub et al., U.S. Pat. No. 5,321,017 to Golub et al., U.S. Pat. No. 5,308,839 to Golub et al., U.S. Pat. No. 5,258,371 to Golub et al., U.S. Pat. No. 4,935,412 to McNamara et al., U.S. Pat. No. 4,704,383 to McNamara et al., U.S. Pat. No. 4,666,897 to Golub et al., and U.S. Pat. No. RE 34,656 to Golub et al. describe the use of non-antimicrobial tetracyclines to treat tissue-destructive conditions, chronic inflammation, bone destruction, cancer and other conditions associated with excess activity of matrix metalloproteinases such as collagenases, gelatinase, and MMP-12 (macrophage metalloelastase).
U.S. Pat. No. 5,773,430 to Simon et al. describes using hydrophobic tetracyclines to inhibit excess leukocyte elastase serine proteinase activity in a biological system.
Tetracyclines are a class of compounds which are particularly well known for their early and spectacular success as antibiotics. Compounds such as tetracycline, sporocycline, etc., are broad spectrum antibiotics, having utility against a wide variety of bacterial and other microbes. The parent compound, tetracycline, has the following general structure: ##STR1##
The numbering system of the multiple ring nucleus is as follows: ##STR2##
Tetracycline, as well as the 5-OH (oxytetracycline, e.g., terramycin.TM.) and 7-Cl (chlorotetracycline, e.g. aureomycin.TM.) derivatives, exist in nature, and are all well known antibiotics. Semisynthetic tetracyclines include, for example, doxycyline, minocycline and methacycline. The use of tetracycline antibiotics, while generally effective for treating infection, can lead to undesirable side effects. For example, the long-term administration of antibiotic tetracyclines can reduce or eliminate healthy flora, such as intestinal flora, and can lead to the production of antibiotic resistant organisms or the overgrowth of yeast and fungi. These significant disadvantages typically preclude treatment regimens requiring chronic administration of these compounds.
Natural tetracyclines may be modified without losing their antibiotic properties, although certain elements of the structure must be retained to do so. A class of compounds has been defined which are structurally related to the antibiotic tetracyclines, but which have had their antibiotic activity substantially or completely extinguished by chemical modification. The modifications that may and may not be made to the basic tetracycline structure were reviewed by Mitscher, L. A., The Chemistry of the Tetracycline Antibiotics, Marcel Dekker, New York (1978), Ch. 6. According to Mitscher, the modification at positions 5-9 of the tetracycline ring system can be made without causing the complete loss of antibiotic properties. However, changes to the basic structure of the ring system, or replacement of substituents at positions 1-4 or 10-12, generally lead to synthetic tetracyclines with substantially less, or essentially no, antibacterial activity.
Chemically modified tetracyclines (CMT's) include, for example, 4-de(dimethylamino)tetracycline (CMT-1), tetracyclinonitrile (CMT-2), 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3), 7-chloro-4-de(dimethylamino)tetracycline (CMT-4), tetracycline pyrazole (CMT-5), 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6), 4-de(dimethylamino)- 12.alpha.-deoxytetracycline (CMT-7), 6-deoxy-5.alpha.-hydroxy-4-de(dimethylamino)tetracycline (CMT-8), 4-de(dimethylamino)-12.alpha.-deoxyanhydrotetracycline (CMT-9), 4-de(dimethylamino)minocycline (CMT-10).
Further examples of tetracyclines modified for reduced antimicrobial activity include the 4-epimers of oxytetracycline and chlorotetracyline (epi-oxytetracycline and epi-chlorotetracycline).
Bisphosphonates include a class of therapeutic preparations which have been used as bone resorption suppressants. U.S. Pat. No. 5,652,227 to Teronen et al. describes using bisphosphonates to reduce degradation of connective tissue matrix protein components which results from excess metalloproteinase activity. U.S. Pat. No. 5,688,120 describes inhibiting alveolar bone resorption using iontophoretic delivery of bisphosphonates to alveolar bone by administering bisphosphonate in a reservoir connected to gingival tissue and passing an electrical current therethrough.
There has been no suggestion to use tetracyclines and bisphosphonates together in combination for the purpose of reducing, inhibiting, and down-regulating excess endogenous proteinase activity and to reduce destruction of tissues, basement membrane and other factors.
It is an object of the invention to provide a combination of compounds to treat subjects susceptible to proteinase related tissue damage and destruction.